The proposed efforts will concentrate on the study of one of the steps in transepithelial sodium transport, namely, Na entry through the apical border. The kinetic approach has been used in the Na entry step to: 1) separate Na binding from translocation, 2) obtain the cationic selectivity of the Na binding site, and 3) describe the interaction between Na, amiloride and other cations in various species of amphibian skin. The biochemical approach used on the Na entry step afforded: 1) amiloride analog studies, and 2) experiments with chemical site-specific reagents. Both of these approaches will continue to be used and expanded, alone or in combination, on various species of amphibian skin with the aims of clarifying the molecular architecture of the Na entry site and of identifying critical protein ligands essential for transport function. Discovery of an irreversible binding agent specific for the Na entry site would be of interest to investigators working in a variety of transport systems. We have found an amiloride analog (bromoamiloride) with such properties and obtained it in labeled 14C form. However, its specific activity was too low for detection by autoradiography. Based on this experience with 14C-bromoamiloride, we will develop and test a new 3H-amiloride analog which inhibits Na entry specifically and irreversibly.